Disrupción de uniones mediadas por Caderinas. Su rol en la apoptosis de las Celulas Granulosas del ovario Porcino

LOMBARDO, DM; MEDINA, J; REVILLA, M.; CAROU, MC; FIORITO, C

Veterinary Research (INVET - Investigaci¨®n Veterinaria)

Universidad de Buenos Aires - BMPress

Lugar: Ciudad de Buenos Aires; Año: 2010 vol. 12 p. 1 - 11

1514-6634

La fisiología normal de los tejidos reproductivos dependera de un apropiado contacto célula-célula, mediado por caderinas, moléculas Ca++ dependientes. El proceso de la atresia dado por apoptosis de las celulas de granulosa (CG), podría estar relacionado con la pérdida de los contactos celulares. El objetivo del trabajo fue establecer la relación entre la disrupción de estas uniones y la apoptosis de las CG. Se realizaron cultivos primarios de CG de ovarios porcinos obtenidos por punción en folículos antrales. Los experimentos fueron realizados a 6, 12 y 24 hs, en concentraciones de 100 y 9 nM de EGTA (quelante exclusivo de Ca++). Se determinó índice de apoptosis (IA) mediante DAPI, TUNEL e ICQ anti Bax y % de viabilidad por exclusión con Azul Tripan. Se observaron manifestaciones evidentes de apoptosis en los diferentes tratamientos con EGTA, sin embargo las diferencias entre el % de viabilidad y los IA por DAPI no son significativas respecto del control. Los resultados observados fueron validados cuali y cuantitativamente por TUNEL e ICQ anti Bax.The normal physiology and the dynamics of the reproductive tissue depends in great measure of an appropriate contact cell to cell, which is mediated by proteins of the cadherins family, molecules Ca dependent such as the E and N Cadherin. In these circumstances, the atresia process for apoptosis of the granulosa cells (CG), it could be related with the loss of this cellular contacts. In this work, the objective was to establish the relationship among the loss of the contacts Ca dependent with the integrity and the apoptosis of the CG. The study model was a cellular primary culture of porcine CG obtained by aspiration of antrals follicles, which were treated with EGTA, to obtain the disruptión of the homophilic juntions. The treatments were made with EGTA 9 nM and 100 nM and without EGTA, both treatment at different times of induction 6, 12 and 24 hours. In each case, the apoptosis index (AI) was determined by DAPI and TUNEL techniques. Also, was carried out a quantitative evaluation by analysis of images of the expression of Bax by inmuno cytochemical. To different concentration of the inductor, the treatments with serum demonstrated a bigger AI to 9 nM (1,27; 2,58 and 1,38 to 6, 12 and 24 hs. The activation was significant in the treatments of 12 hs with serum respect to the control. To concentrations of 100 nM the AI didn´t demonstrate significant differences of activation. The quantitative determination of the expression of Bax and for ICQ validates the previous results.